The present invention relates to methods and media for the storage of blood platelets, and more particularly to methods and media for storing those platelets at temperatures from about 18.degree.-30.degree. C., preferably in the range from about 18.degree.-30.degree. C., preferably in the range of about 20.degree.-24.degree. F.. and even more preferably at about 22.degree. C.
Platelets are obtained as a by-product from whole blood donations and from plateletpheresis procedures. Typically, they are now stored in their own plasma within a plastic container whose walls are permeable to atmospheric gases. The plasma associated with these platelets normally contains all the ingredients of normal plasma, plus citrate, which is added as an anti-coagulant, and dextrose at 5 times the physiologic level. The increased dextrose is added for the benefit of red cells which require it during storage, and is generally accepted to be required for platelet storage as well.
In routine blood banking practice, donations of a unit of blood (450 ml into 67.5 anti-coagulant) are processed by centrifugation into three fractions--red cells, plasma, and platelets. The volume of packed red cells from a unit is approximately 180 ml with a remaining volume of plasma and anti-coagulant of about 337.5 ml. As used in the remainder of this application, the term "plasma" includes any anti-coagulant which has been added thereto at the time of its initial collection. The red cells (referred to as packed red cells) are typically suspended in approximately 47.5 ml of plasma. Platelets are suspended in approximately 50 ml of plasma. This platelet containing product is typically referred to as platelet concentrate. The remaining 240 ml of plasma is frozen as fresh frozen plasma.
Recent advances have allowed blood banks to store platelet concentrates at 22.degree. C. for five days. See Murphy et al, "Improved Storage of Platelets for Transfusion in a New Container", Blood 60(1):194-200 (1982); Simon et al, "Extension of Platelet Concentrate Storage", Transfusion, 23(3):207-212 (1983). The extended storage of platelets at 22.degree. C. provides the flexibility to allow inventory planning so that platelets can be available at widely dispersed sites when needed. However, the above described storage procedure requires that platelets be suspended in 50 ml plasma which is then infused into the patient along with the platelets.
There are disadvantages to storing platelets in large volumes of plasma which are then infused into a recipient. Diseases may be transmitted by plasma infusion. It is certain that hepitatis-B and nonA, nonB-hepitatis may be transmitted by such plasma infusions. It has also now been reported that the newly recognized acquired immunodeficiency syndrome (AIDS) may be transmitted through plasma infusion. Patients may also exhibit allergic reactions to plasma which are at least annoying, and occasionally fatal. Furthermore, plasma is valuable because it can be fractionated into its components such as albumin and coagulation factors for treatment of specific patients. A milliliter of plasma is worth at least about 4-5 cents. Therefore, the plasma used to suspend platelets is worth as much as $2.50 per unit. Since 4 million units of platelets are administered yearly in the United States, the use of plasma as a storage medium for platelets may waste up to $10 million annually of plasma. Furthermore, the use of platelets in the United States has been increasing as a general trend.
A great deal is known about human platelet cells. General papers describing techniques, materials and methods for storage of blood platelets are described by Murphy et al. in "Improved Storage of Platelets for Transfusion in a New Container", Blood 60(1), July, 1982; by Murphy in "The Preparation and Storage of Platelets for Transfusion", Mammer, Barnhart, Lusher and Walsh, PJD PUblications Ltd., Westbury, N.Y. (1980); by Murphy in "Platelet Transfusion", Progress in Hemostasis and Thrombosis, Vol. III, Edited by Theodore H. Spaet, Grune and Stratton, Inc. (1976); and by Murphy et al. in "Platelet Storage at 22.degree. C.: Role of Gas Transport Across Plastic Containers in Maintenance of Viability", Blood 46(2):209-218 (1975) each of which publications is hereby incorporated by referenceaas if fully set forth herein.
There are, of course, many culture media and/or physiologic solutions which are deemed acceptable for use in maintaining and/or culturing vertebrate cells. Such solutions include Earle's solution (a tissue culture medium); Fonio's solution (used for stained smears of blood platelets); Gey's solution (for culturing animal cells); Hank's solution (for culturing animal cells); Heyem's solution (a blood diluent used prior to counting red blood cells); Krebs-Ringers solution (a modification of Ringers solution prepared by mixing NaCl, KCl, CaCl.sub.2, MgSO.sub.4, and phosphate buffer, pH 7.4); lactated Ringers solution (containing NaCl, sodium lactate, CaCl.sub.2 (dihydrate) and KCl in distilled water); Locke's solution (for culturing animal cells); Locke-Ringers solution (containing NaCl, CaCl.sub.2, KCl, MgCl.sub.2, NaHCO.sub.3, glucose and water); Ringers solution (resembling blood serum and its salt constituents, containing 8.6 grams of NaCl, 0.3 gms of KCl and 0.33 gms of CaCl in each thousand milliliters of distilled water, used typically for burns and wounds or used in combination with naturally occurring body substances, e.g., blood serum, tissue abstracts and/or more complex chemically defined nuclear solutions for culturing animal cells); and Tyrode's solution (a modified Locke's solution). See Stedman's Medical Dictionary, pp. 1300-1301, Williams and Wilkins, Baltimore, Md. (1982).
The maintenance and/or culturing of live vertebrate cells creates different problems depending upon the particular type of cells employed. It is known, for example, that blood cells, such as platelets, have many metabolic properties similar to those of certain other types of cells. In Blood 30:151 (1967), for example, it is suggested that the metabolic properties of blood cells are similar in some respects to those of tumor cells. On the other hand, there exists a considerable body of prior art directed specifically to the problem of storing stable suspensions of blood cells, such as blood platelets. Prior work on the storage of blood platelets, has shown that the duration of platelet storage is limited by the continuing production of lactic acid from dextrose by the platelets. Although this provides energy for the platelets, the lactic acid acidifies the medium, which acidity eventually destroys the platelets. It has also been shown that platelets consume oxygen during storage for energy production, the end product of which process is a gas, CO.sub.2 which, unlike lactic acid, can leave the container through the plastic walls in which it is normally stored. The production of CO.sub.2 does not acidify the storage medium for these platelets. In addition to the glycolysis of dextrose, fatty acids and amino acids typically present in the plasma may be used as substrates for oxidative metabolism of stored platelet cells.
Various techniques are disclosed in the patent literature relating to the fractionation of blood into useful end products. In British Patent No. 1,283,273, a method and apparatus for separating plasma from whole blood to produce a plasma end product is disclosed wherein the remaining blood fractions are returned to a donor. In U.S. Pat. No. 4,269,718 (Persidsky) a method of centrifugal separation of platelets from blood using saline as a washing and displacing solution is disclosed. In Prandi, U.S. Pat. No. 4,387,031, a composition is disclosed for separating erythrocytes and plasma in blood.
The patent literature also discloses various preservative or culture media which are disclosed as being useful in conjunction with the storage of platelets. In U.S. Pat. No. 4,390,619 (Harmening-Pittiglio) an ion-exchange resin for slow release of phosphate buffer is disclosed. This patent contains an extensive discussion of oxidative phosphorylation and glycolysis mechanisms of platelets, and the relationship thereof to platelet storage. In U.S. Pat. No. 2,786,014 (Tullis) and U.S. Pat. No. 3,629,071 (Sekhar) various glucose and electrolyte containing platelet storage media are disclosed which are intended for use in the storage of platelets at temperatures in the range of 4.degree.-5.degree. C. Similarly, U.S. Pat. No. 4,152,,208 discloses a method and medium for storing stabilized leucocytes which are disclosed as being stored at temperatures from about 4.degree. C. to about 30.degree. C., which are kept in a basic salt solution or a minimum essential medium which sustains the viability of the leucocytes in the blood. In U.S. Pat. No. 4,267,269 (Grode et al.) a red cell storage solution is disclosed containing adenine, glucose or fructose, sodium chloride and mannitol. U.S. Pat. No. 3,814,687 (Ellis et al.), U.S. Pat. No. 3,850,174 (Ayes) and the aforementioned British No. 1,283,273 patent generally relate to the separation of formed elements from plasma in blood fractionation processes. In U.S. Pat. No. 4,152,208 (Guirgis) a variety of leucocyte preservative solutions and the use of such solutions in blood fractionation procedures is disclosed, including Eagles' MEM (columns 3 and 4) containing inter alia, glutamine, leucine, isoleucine, phenylalanine, tyrosine, phosphates and sodium and potassium chlorides, for use in conjunction with anti-coagulants such as sodium citrate. See also U.S. Pat. No. 4,205,126 (Cartaya). U.S. Pat. No. 3,753,357 (Schwartz) broadly disclosed various preservative or culture media useful in conjunction with the storage of blood cells.
In "Platelet Size and Kinetics in Hereditary and Acquired Thrombocytopenia" by Murphy et al., New England Journal of Medicine 286: 499-504 (Mar. 9, 1972), a method is disclosed for determining platelet volume wherein blood samples are drawn, mixed with acid-citrate-dextrose, and centrifuged to obtain platelet-rich plasma. The platelet-rich plasma is then diluted in Isoton or an isotonic buffer of sodium chloride, potassium chloride and sodium phosphate, and adjusted to a pH of 7.4 with hydrochloric acid. A Coulter counter is then used to determine platelet count and platelet volume in these samples. As disclosed at page 4 of this paper, incubation of the platelets may also be carried out with radiolabelled chromium in a Ringer-Citrate-Dextrose Solution for one hour, in accordance with the technique of Abrahamsen.
In Abrahamsen, "A Modification of the Technique for .sup.51 Cr-Labeling of Blood Platelets Giving Increased Circulating
Platelet Radioactivity", Scand. J. Haemat. 5:53-63 (1968) the influence of temperature, pH, incubation medium and inoculation time on the uptake of radiolabelled chromium by platelets is discussed. As disclosed at page 54 of this reference, in vitro studies were conducted on platelets which were separated from platelet-rich ACD-plasma (obtained from whole blood donations) by centrifugation. Different samples of these platelets were resuspended in equal volumes of incubation media and "left for half an hour at the temperature selected for the experiment". Among the media tested were saline-citrate-dextrose, Ringers-citrate-dextrose and Ringers-citrate. Identical amounts of radiochromium were then added to these suspensions and incubation continued until it was stopped after various periods of time by adding ascorbic acid. See FIG. 1 at page 55 of Abrahamsen. In vivo studies were also conducted wherein ACD-platelets were incubated at 20 ml. of Ringers-Citrate-Dextrose Solution for various periods of time at the temperature and with the concentration of radiocromium for the selected experiment. In these in vivo studies, samples of the incubated platelets were reinfused into the patient, and recovered to determine, for example, platelet half-lifetime.
In addition to these studies, Abrahamsen reports his concern that certain factors which increase platelet uptake of radiochromium "might induce irreversible platelet damage with reduced recovery and shortening of platelet survival." Abrahamsen reports that platelet recovery and survival time were evaluated using various incubation procedures in Ringers-Citrate-Dextrose which were compared with the results after incubation in ACD plasma in the same subject. Abrahamsen reports:
Platelet recovery and survival were not affected when platelets were incubated in Ringer-Citrate-Dextrose for one-half, one and two hours . . . or after increasing chromate dosage." PA0 "According to these findings, incubation in Ringer-Citrate at room temperatures seems preferable. Even a platelet uptake of .sup.51 CR after incubation for one hour or less is nearly identical in saline and Ringer-Citrate-Dextrose is preferred as incubation medium." See Abrahamsen at page 59.
Abrahamsen concludes:
In addition to the above, please refer to U.S. Pat. No. 4,447,415 (Rock et al.) and to Adams GA et al. "Is Plasma Really Required for Platelet Storage? ", Abstract No. 628 at page 175a Blood, Nov., 1982.
As discussed hereinafter, one of the preferred embodiments of the present invention utilizes a synthetic platelet storage medium to which glutamine has been added. Although not related to the storage or culturing of blood cells, the following publications do disclose that glutamine may be a substrate for oxidative phosphorylation in a certain type of cancer cells, namely, HeLa cells. See "The Continuous Growth of Vertebrate Cells in the Absence of Sugar" by Wice et al., Journal of Biological Chemistry, 256(15): 7812-7819 (1981); "The Pentose Cycle", by Reitzer et al., Journal of Biological Chemistry, 255(12): 5616-5626 (1980); and "Evidence that Glutamine, Not Sugar is the Major Energy Source for Cultured HeLa Cells", by Reitzer et al., Journal of Biological Chemistry, 254(8):2669-2676 (1979). See also Kuchler, Biochemical Methods in cell Culture and virology Halstead Press at page 83, 87-88, (1977).
Notwithstanding the considerable work conducted in this area, a need still exists for a simple safe, inexpensive method for storing human blood platelets in viable condition while increasing the amount of blood plasma which is freed for other uses.